Fig 1.
3D platform for spheroid culture.
(A) Spheroid culture process onto the non-adherent concave bed in a 96-well plate. (B) peeled off PDMS layer from a well.
Fig 2.
5FU-CUR-PIP-HSA-NPs characterization.
(A) SEM image and (B) particle size distribution of 5FU-CUR-PIP-HSA-NPs. (C) DSC thermograms of 5FU, PIP, CUR, HSA, and 5FU-CUR-PIP-HSA-NPs. (D) In vitro release patterns of the three drugs from 5FU-CUR-PIP-HSA-NPs in different buffers at 37°C. Each point represents the average of three independent experiments, and error bars show the SD.
Fig 3.
(A) Sequential images of spheroid culture with the initial cell density of 30,000 cells/well. The images were taken with 4X magnification by an optical microscope. Scale bars = 200 μm. (B) SEM images of the generated MDA-MB-231spheroid with the initial density of 40,000 cells per spheroid on day 7 with different magnifications.
Fig 4.
Tumor spheroid characterization.
(A) Brightfield image of the tumor spheroid and the same image with pathophysiological concentric zones. Scale bar = 100 μm (B) Fluorescent image of live/dead assay on a tumor spheroid using FDA (green) and PI (red)). Scale bar = 200 μm. (C) Flow cytometric annexin V/PI analysis of MDA-MB-231 spheroids after 4 days. Quadrant assignment: Lower left = viable cells; lower right = early apoptotic cells; upper right = late apoptotic cells; upper left = necrotic cells.
Fig 5.
Penetration of therapeutics/nanomedicine (5FU-CUR-PIP-HSA-NPs) in MDA-MB-231 cancer cells in 2D and 3D conditions.
In vitro uptake of (A) free drugs combination and (B) 5FU-CUR-PIP-HSA-NPs in 2D monolayer cancer cells at the CUR/5FU/PIP concentration of 50/26.8/7.4 μg/ml after 4 h. Scale bars = 50 μm. In vitro uptake of (C) free drugs combination and (D) 5FU-CUR-PIP-HSA-NPs with different concentrations and incubation times in 3D tumor spheroids. The green fluorescence represents CUR. Scale bars = 200 μm.
Fig 6.
Cytotoxicity and anti-metastatic activity of 5FU-CUR-PIP-HSA-NPs on MDA-MB-231 cancer cells.
(A) Anticancer activity of free drugs combination and 5FU-CUR-PIP-HSA-NPs on MDA-MB-231 monolayer (2D) and spheroids (3D) after the 48 h treatment. Each represented data point is calculated as the mean ±SD of four experiments. (B) Migration inhibition of metastatic MDA-MB-231 cells by wound healing assay. After the scratch creation, cells were treated with 5FU-CUR-PIP-HSA-NPs. After 24 h, the wound’s gap became completely covered in the control group, meanwhile, the migration of metastatic cells was suspended using a low concentration of the NPs. Scale bars = 50 μm.